Human cytokines

ABSTRACT

The present invention provides novel human cytokines (designated individually as NHC-1 and NHC-2, and collectively as NHC) and polynucleotides which identify and encode NHC. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding NHC and a method for producing NHC. The invention also provides for use of NHC and agonists, antibodies, or antagonists specifically binding NHC, in the prevention and treatment of diseases associated with expression of NHC. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding NHC for the treatment of diseases associated with the expression of NHC. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding NHC.

[0001] This application is a divisional application of U.S. applicationSer. No. 09/782,142, filed Feb. 12, 2001, entitled HUMAN CYTOKINES,which is a divisional application of U.S. application Ser. No.08/792,013, filed Jan. 31, 1997, now U.S. Pat. No. 6,204,021, issuedMar. 20, 2001, entitled DNA ENCODING A CYTOKINE, and all of which arehereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof novel human cytokines and to the use of these sequences in thediagnosis, prevention, and treatment of cancers, inflammation, allograftrejection, neurodegenerative diseases, and conditions affectingpregnancy, growth and development

BACKGROUND OF THE INVENTION

[0003] Cytokines are active in cell proliferation and differentiationand affect such activities as leukocyte migration and function,hematopoietic cell numbers, temperature regulation, acute response toinfections, tissue remodeling and cell survival. Since cytokines areproduced in groups and in patterns characteristic of the particularstimulus or disease, studies using antibodies or other drugs that modifythe activity of a particular cytokine are beginning to elucidate theroles of individual cytokines in pathology and physiology. For purposesof example, two cytokines which are rapidly expressed in response tofibroblast growth factor (FGF) and inflammatory response (AIF-1),respectively, will be described.

[0004] FIN14 is an inducible gene which was expressed in cellstransfected with a genomic fragment of the FGF-4, a growth factor knownto play a crucial role in mouse and Xenopus embryonic growth anddevelopment (Feldman, B et al. (1995) Science 267:246-9; Amaya, E. etal. (1991) Cell 66:257-70). The transcript for FIN 14 was originallyisolated using subtractive hybridization between the transformed 15 cellline and untransfomed mouse NIH3T3 cells, the line from which A15 wasderived. FIN 14 is maximally induced 12-18 hours after FGF-4 treatmentand is considered to be a delayed or late response transcript whencompared to intermediate-early response genes such as transcriptionfactors. Further studies showed that FIN14 maps to the distal arm ofmouse chromosome 6 and is also expressed in response to mitogenicstimulation (serum; Guthridge, M. A. (1996) Oncogene 12:1267-78).

[0005] AIF-1, allograft inflammatory factor-1, was first cloned andcharacterized from rat cardiac allografts. AIF-1 is a 17 kD hydrophillicprotein which is selectively expressed in macrophages and neutrophils,and when stimulated by T-cell interferon-gamma, AIF-1 expressionincreases six-fold. AIF-1 transcripts are lacking in cardiac syngraftsand in host hearts, and transcript levels can be reduced in allograftsby introducing CTLA-4 immunoglobulin (Utans, U. et al. (1995) J. Clin.Invest. 95:2954-62).

[0006] AIF-1 has 77% overall amino acid identity with IBA1; however,most of the variability is in the first 9 and in the last 20 residues.Both of these molecules share a calcium binding domain,D₅₈LNGNGDIDIMSL₇₀. (Imai, Y et al. (1996) Biochem Biophys. Res. Comm.224:855-862) report that IBA1 is highly expressed in spleen, testes, andmicroglia where the protein may mediate calcium signaling.

[0007] The discovery of novel cytokines and the molecules encoding themprovides a means to investigate cell proliferation, leukocyte migrationand function, response to infections and tissue remodeling under normaland disease conditions. Such novel cytokines satisfy a need in the artby providing new compositions useful in diagnosing and treating cancers,inflammation, allograft rejection, neurodegenerative diseases, andconditions affecting pregnancy, growth and development

SUMMARY OF THE INVENTION

[0008] The present invention features two novel cytokines, designatedindividually as NHC-1 and NHC-2 and collectively as NHC, andcharacterized as having similarity to mouse FIN14 and AIF-1,respectively

[0009] Accordingly, the invention features substantially purified NHCproteins NHC-1 and NHC-2 having the amino acid sequences shown in SEQ IDNO:1 and SEQ ID NO:4, respectively.

[0010] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode NHC proteins--NHC-1 and NHC-2. In aparticular aspect, the polynucleotides are the nucleotide sequences ofSEQ ID NO:2 and SEQ ID NO:5, respectively.

[0011] The invention also features a polynucleotide sequence comprisingthe complement of SEQ ID NO:2 and SEQ ID NO:5, or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2 and SEQ ID NO:5.

[0012] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode NHC. Thepresent invention also features antibodies which bind specifically toNHC, and pharmaceutical compositions comprising substantially purifiedNHC. The invention also features the use of agonists and antagonists ofNHC.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIG. 1 shows the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of NHC-1. The alignment was produced usingMACDNASIS PRO software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

[0014]FIG. 2 shows the amino acid sequence alignment between NHC-1 (SEQID NO:1) and FIN14 (GI 1353711; SEQ ID NO:3).

[0015]FIGS. 3A and 3B show the hydrophobicity plots (MACDNASIS PROsoftware) for NHC-1 (SEQ ID NO:1) and FIN14 (GI 1353711; SEQ ID NO:3),respectively; the positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

[0016]FIGS. 4A and 4B show the northern analysis of NHC-1 produced usingthe LIFESEQ® database (Incyte Pharmaceuticals, Palo Alto, Calif.).

[0017]FIG. 5 shows the amino acid sequence (SEQ ID NO:4) and nucleicacid sequence (SEQ ID NO:5) of NHC-2.

[0018]FIG. 6 shows the amino acid sequence alignments among NHC-2 (SEQID NO:4), human AIF-1 (GI 1229022; SEQ ID NO:6), and rat IBA1 (GI1514969; SEQ ID NO:7).

[0019]FIGS. 7A and 7B show the northern analysis for NHC-2 producedusing the LIFESEQ® database.

DESCRIPTION OF THE INVENTION

[0020] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0021] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0022] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0023] Definitions

[0024] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0025] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0026] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0027] NHC, as used herein, refers to the amino acid sequences ofsubstantially purified NHC obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant. “Consensus”, as used herein, refers to anucleic acid sequence which has been resequenced to resolve uncalledbases, or which has been extended using XL-PCR (Applied Biosystems,Foster City, Calif.) in the 5′ and/or the 3′ direction and resequenced,or which has been assembled from the overlapping sequences of more thanone Incyte clone using the GELVIEW Fragment Assembly system (GCG,Madison, Wis.), or which has been both extended and assembled.

[0028] A “variant” of NHC, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0029] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0030] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0031] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0032] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic NHC, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0033] The term “agonist”, as used herein, refers to a molecule which,when bound to NHC, causes a change in NHC which modulates the activityof NHC. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to NHC.

[0034] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to NHC, blocks or modulates the biological orimmunological activity of NHC. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to NHC.

[0035] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of NHC. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of NHC.

[0036] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of NHCor portions thereof and, as such, is able to effect some or all of theactions of cytokines.

[0037] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding NHC or the encoded NHC.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0038] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0039] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0040] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0041] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C_(O)t or R_(O)tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0042] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0043] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0044] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0045] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tim As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0046] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0047] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human NHC-1 and fragments thereof.

[0048] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0049] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0050] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0051] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding NHC orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0052] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 or SEQ ID NO:5 by northern analysisis indicative of the presence of mRNA encoding NHC in a sample andthereby correlates with expression of the transcript from thepolynucleotide encoding the protein.

[0053] “Alterations” in the polynucleotide of SEQ ID NO:2 or SEQ ID NO:5as used herein, comprise any alteration in the sequence ofpolynucleotides encoding NHC including deletions, insertions, and pointmutations that may be detected using hybridization assays. Includedwithin this definition is the detection of alterations to the genomicDNA sequence which encodes NHC (e.g., by alterations in the pattern ofrestriction fragment length polymorphisms capable of hybridizing to SEQID NO:2 or SEQ ID NO:5), the inability of a selected fragment of SEQ IDNO:2 or SEQ ID NO:5 to hybridize to a sample of genomic DNA (e.g., usingallele-specific oligonucleotide probes), and improper or unexpectedhybridization, such as hybridization to a locus other than the normalchromosomal locus for the polynucleotide sequence encoding NHC (e.g.,using fluorescent in situ hybridization (FISH) to metaphase chromosomesspreads).

[0054] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind NHCpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe translation of mRNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0055] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0056] The Invention

[0057] The invention is based on the discovery of novel human cytokines(NHC-1 and NHC-2, collectively referred to as NHC), the polynucleotidesencoding NHC, and the use of these compositions for the diagnosis,prevention, or treatment of cancers, inflammation, allograft rejection,neurodegenerative diseases, and conditions affecting pregnancy, growthand development.

[0058] Nucleic acid sequence encoding the human NHC-1 of the presentinvention were first identified in Incyte Clone 1431384 from an ileumlibrary of a patient with irritable bowel syndrome (SINTBST01) through acomputer-generated search for amino acid sequence alignments. Aconsensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences (cDNA library fromwhich derived): Incyte Clones 1431384 (SINTBST01), 764682 (LUNGNOT04),and 1813445 (PROSTUT12).

[0059] Nucleic acid sequence encoding the human NHC-2 of the presentinvention were first identified in Incyte Clone 815614 from an ovariantumor cDNA library (OVARTUT01) through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:5, wasderived from the following overlapping and/or extended nucleic acidsequences (cDNA library from which derived): Incyte Clones 815614(OVARTUT01), 690707 (LUNGTUT02), 292061 (TMLR3DT01), and 522938(MMLR2DT01).

[0060] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, and shown in FIG. 1.NHC-1 is 69 amino acids in length and contains a potentialmyristoylation site at G₇. As shown in FIG. 2, NHC-1 has chemical andstructural homology with FIN14 from mouse fibroblast cells (GI 1353711;SEQ ID NO:3). In particular, NHC-1 shares 76% identity with FIN14. Asillustrated by FIG. 3A and 3B, NHC-1 and FIN14 have rather similarhydrophobicity plots although the signal sequence of NHC-1 is morehydrophobic. Northern analysis (FIGS. 4A and 4B) shows the expression ofNHC-1 in libraries from immortalized cell lines and cancerous tissues(44/111) and from cells and tissues associated with inflammation(29/111). In addition, the presence of NHC-1 in several libraries fromneonates, infants or children below the age of 10 (14/111) suggests thatNHC-1 has a role in growth and development.

[0061] In another embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:4, and shown in FIG.5.NHC-2 is 147 amino acids in length and contains several potentialphosphorylation sites at Y₃₇, S₃₈, S₃₉, S₄₅, S₆₉, T₈₂, and S₁₀₂, andpotential myristoylation sites at G₁₄, G₇₈, and G₉₆. As shown in FIG. 6,NHC-2 has chemical and structural homology, including the calciumbinding domain from D₅₈ to L₇₀, with AIF1 (GI 1229022; SEQ ID NO:6) andrat IBA1 (GI 1514969; SEQ ID NO:7). In particular, NHC-2 shares share89% and 86% identity with AIF1 and rat IBA1, respectively. Northernanalysis (FIGS. 7A and 7B) reveals the expression of NHC-2 in librariesfrom immortalized cell lines and cancerous tissues (20/61) and fromcells and tissues associated with inflammation (19/61). In addition, thepresence of NHC-2 in several brain and neural libraries suggests thatNHC-2 has a role in neurodegnerative diseases.

[0062] The invention also encompasses NHC variants. A preferred NHCvariant is one having at least 80%, and more preferably 90%, amino acidsequence similarity to the NHC amino acid sequences (SEQ ID NO:1 and SEQID NO:4). A most preferred NHC variant is one having at least 95% aminoacid sequence similarity to SEQ ID NO:1 and SEQ ID NO:4.

[0063] The invention also encompasses polynucleotides which encode NHC.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of NHC can be used to generate recombinant molecules whichexpress NHC. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid of SEQ ID NO:2 and SEQ IDNO:5, as shown in FIGS. 1 and 5, respectively.

[0064] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding NHC, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring NHC, and all such variations are to beconsidered as being specifically disclosed.

[0065] Although nucleotide sequences which encode NHC and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NHC under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NHC or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding NHC and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0066] The invention also encompasses production of DNA sequences, orportions thereof, which encode NHC and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding NHC or any portion thereof.

[0067] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2 and SEQ ID NO:5, under variousconditions of stringency. Hybridization conditions are based on themelting temperature (Tm) of the nucleic acid binding complex or probe,as taught in Wahl, G. M. and S. L. Berger (1987; Methods Enzymol.152:399-407) and Kimmel, A. R. (1987; Methods Enzymol. 152:507-11), andmay be used at a defined stringency.

[0068] Altered nucleic acid sequences encoding NHC which are encompassedby the invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent NHC. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentNHC. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of NHC is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

[0069] Also included within the scope of the present invention arealleles of the gene encoding NHC. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0070] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE DNA Polymerase (US Biochemical Corp,Cleveland, Ohio), Taq polymerase (Applied Biosystems), thermostable T7polymerase (Amersham, Chicago, Ill.), or combinations of recombinantpolymerases and proofreading exonucleases such as the ELONGASEamplification (GIBCO/BRL, Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton MICROLAB 2200 (Hamilton,Reno, N.Y.), Peltier thermal cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Applied Biosystems).

[0071] The nucleic acid sequences encoding NHC may be extended utilizinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0072] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 primer analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0073] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore peforming PCR.

[0074] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0075] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0076] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled devise camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Applied Biosystems) and the entire process fromloading of samples to computer analysis and electronic data display maybe computer controlled. Capillary electrophoresis is especiallypreferable for the sequencing of small pieces of DNA which might bepresent in limited amounts in a particular sample.

[0077] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode NHC, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of NHC in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressNHC.

[0078] As will be understood by those of skill in the art, it may beadvantageous to produce NHC-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0079] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to altersequences encoding NHC for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, or to introduce mutations, and so forth.

[0080] In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding NHC may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of NHC activity, it may be useful toencode a chimeric NHC protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a sequence encoding NHC and theheterologous protein sequence, so that NHC may be cleaved and purifiedaway from the heterologous moiety.

[0081] In another embodiment, sequences encoding NHC may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of NHC, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431Apeptide synthesizer (Applied Biosystems).

[0082] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of NHC, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0083] In order to express a biologically active NHC, the nucleotidesequences encoding NHC or functional equivalents, may be inserted intoappropriate expression vectors, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0084] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding NHCand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0085] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding NHC. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or PBR322 plasmids); or animal cell systems.

[0086] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL), and the like, may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding NHC,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0087] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for NHC. For example, whenlarge quantities of NHC are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding NHC may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. PGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0088] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0089] In cases where plant expression vectors are used, the expressionof a sequence encoding NHC may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter, J.et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructscan be introduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, for example, Hobbs, S. orMurry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y.; pp. 191-196).

[0090] An insect system may also be used to express NHC. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding NHCmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of NHC will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which NHC may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0091] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding NHC may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing NHC in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0092] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding NHC. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding NHC, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0093] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0094] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress NHC may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0095] Any number of selection systems may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻ or aprt⁻ cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad.Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131). .

[0096] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding NHC isinserted within a marker gene sequence, recombinant cells containingsequences encoding NHC can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding NHC under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

[0097] Alternatively, host cells which contain sequences encoding andexpressing NHC may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA-DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

[0098] The presence of polynucleotide sequences encoding NHC can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding NHC. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding NHC to detect transformantscontaining DNA or RNA encoding NHC. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

[0099] A variety of protocols for detecting and measuring the expressionof NHC, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson NHC is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0100] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding NHCinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, sequencesencoding NHC, or any portion thereof, may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T17, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits from Pharmacia & Upjohn(Kalamazoo, Mich.); Promega (Madison, Wis.); and U.S. Biochemical Corp.(Cleveland, Ohio). Suitable reporter molecules or labels, which may beused, include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0101] Host cells transformed with nucleotide sequences encoding NHC maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNHC may be designed to contain signal sequences which direct secretionof NHC through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding NHC tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAG extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and NHC may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingNHC and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying NHC from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

[0102] In addition to recombinant production, fragments of NHC may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Aim Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431Apeptide synthesizer (Applied Biosystems). Various fragments of NHC maybe chemically synthesized separately and combined using chemical methodsto produce the full length molecule.

[0103] Therapeutics

[0104] NHC-1 has chemical and structural homology with FIN14 from mousefibroblast cells. As shown in FIG. 4A and 4B, NHC-1 is expressed inimmortalized or cancerous cells and tissues such as leukemias andlymphomas and cancers of the bladder, brain, breast, colon, gallbladder, heart, kidney, lung, ovary, pancreas, parathyroid, prostate,skull, testicles, and thyroid. It is also expressed in cells and tissuesinvolved in inflammation such as adenoid, bronchial epithelium,granulocytes, leukocytes, lymphocytes, macrophages, placenta, spleen,and thymus, and in tissues from patients with diagnosed autoimmunediseases such as asthma, Crohn's disease, emphysema, multiple sclerosis,and rheumatoid arthritis. Of particular note is the expression of NHC-1in placenta and fetal tissues such as brain, heart, kidney, lung, andspleen. Based on the 76% amino acid identity between NHC-1 and FIN14,and the expression of NHC in proliferating and migrating cells andtissues, NHC-1 has an active role in cancers, inflammation, andconditions affecting pregnancy, growth and development.

[0105] Therefore, in one embodiment, NHC-1 or a fragment or derivativethereof may be added to cells obtained from a subject or a cell line tostimulate cell proliferation. This embodiment is particularly useful forex vivo therapies involving bone marrow cells, and for regeneratingtissues or organs for transplantation such as kidney, liver, pancreas,and spleen.

[0106] In another embodiment, a vector capable of expressing NHC-1, or afragment or a derivative thereof, may be added to cells to stimulatecell proliferation as described above.

[0107] In another embodiment, agonists which stimulate or prolong theactivity of NHC-1 may be added to cells to stimulate cell proliferationas described above.

[0108] In another embodiment, antagonists or inhibitors of NHC-1 may beadministered to a subject to treat or prevent various types of cancer.Types of cancers may include, but are not limited to, the cancers listedabove.

[0109] In another embodiment, antagonists or inhibitors of NHC-1 may beadministered to a subject to treat or prevent various types ofinflammation. Types of inflammation may include, but are not limited to,inflammation caused by viral, bacterial, fungal, helminthic or protozoalinfection; trauma; autoimmune diseases such as hemolytic anemia,arteriosclerosis, asthma, biliary cirrhosis, cystic fibrosis, diabetes,glomerulonephritis, hyperthyroidism, pulmonary fibrosis, systemic lupus,leukemia, myasthenia gravis, osteoporosis, pancreatitis, Sjögren'ssyndrome, scleroderma, and any other conditions specifically listedabove.

[0110] In another embodiment, antagonists or inhibitors of NHC-1 may beadministered to a subject to prevent pregnancy by preventing cellproliferation which results in the implantation of the embryo in theuterus.

[0111] In another embodiment, a vector expressing antisense of thepolynucleotide encoding NHC-1 may be administered to a subject to treator prevent various types of cancer. Types of cancers may include, butare not limited to, the cancers listed above.

[0112] In another embodiment, a vector expressing antisense of thepolynucleotide encoding NHC-1 may be administered to a subject to treator prevent various types of inflammation. Types of inflammation mayinclude, but are not limited to, those listed above.

[0113] NHC-2 has chemical and structural homology with AIF-1. As shownin FIGS. 7A and 7B, NHC-2 is expressed in immortalized or cancerouscells and tissues, particularly cancers of the bladder, brain, breast,intestine, lung, ovary, pancreas, prostate, skull, and stomach. It isalso expressed in bone marrow, granulocytes, leukocytes, lymphocytes,macrophages, mast cells, and fetal spleen; in cells and tissues frompatients with diagnosed autoimmune diseases such as Crohn's disease,multiple sclerosis, and rheumatoid arthritis; and in brain or neuralcells and tissues. Based on the amino acid identity among NHC-2, AIF-1,and IBA1 and the expression of NHC-2 in proliferating and migratingcells and tissues, NHC-2 has an active role in allograft rejection,cancers, inflammation, and neurodegenerative conditions affecting thebrain and nervous system

[0114] In one embodiment, antagonists or inhibitors of NHC-2 may beadministered to a subject to treat or prevent allograft rejection.

[0115] In another embodiment, antagonists or inhibitors of NHC-2 may beadministered to a subject to treat or prevent various types of cancer.Types of cancers may include, but are not limited to, the cancers listedabove.

[0116] In another embodiment, antagonists or inhibitors of NHC-2 may beadministered to a subject to treat or prevent various types ofinflammation. Types of inflammation may include, but are not limited to,inflammation caused by viral, bacterial, fungal, or protozoal infection;trauma; autoimmune diseases such as hemolytic anemia, arteriosclerosis,asthma, biliary cirrhosis, cystic fibrosis, diabetes,glomerulonephritis, hyperthyroidism, pulmonary fibrosis, systemic lupus,leukemia, myasthenia gravis, osteoporosis, pancreatitis, Sjögren'ssyndrome, scleroderma, and those conditions specifically listed above.

[0117] In another embodiment, antagonists or inhibitors of NHC-2 may beadministered to a subject to treat or prevent various types ofneurodegenerative disease. Types of neurodegenerative disease includebut are not limited to, Alzheimier's disease, amylotropic lateralsclerosis, Huntington's disease, Parkinson's disease, epilepsy, andDown's syndrome.

[0118] In another aspect, antibodies which are specific for NHC-1 orNHC-2 may be used directly as an antagonists, or indirectly as targetingor delivery mechanisms for bringing a pharmaceutical agent to cells ortissue which express NHC.

[0119] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0120] Antagonists or inhibitors of NHC may be produced using methodswhich are generally known in the art. In particular, purified NHC may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind NHC.

[0121] The antibodies may be generated using methods that are well knownin the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0122] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith NHC or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0123] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to NHC have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of NHC amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0124] Monoclonal antibodies to NHC may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J Immunol. Methods 81:31-42; Cote, R. J. et al.(1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1985)Mol Cell Biol. 62:109-120.

[0125] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-55; Neuberger, M. S. et al. (1984) Nature312:604-8; Takeda, S. et al. (1985) Nature 314:452-4). Alternatively,techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce NHC-specificsingle chain antibodies. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated by chain shuffling fromrandom combinatorial immunoglobulin libraries (Burton D. R. (1991) Proc.Natl. Acad. Sci. 88:11120-3).

[0126] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-37; Winter, G. et al. (1991) Nature 349:293-9).

[0127] Antibody fragments which contain specific binding sites for NHCmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-81).

[0128] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between NHC and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NHC epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

[0129] In another embodiment of the invention, the polynucleotidesencoding NHC, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding NHC may be used in situations in which it wouldbe desirable to block the transcription of mRNA. In particular, cellsmay be transformed with sequences complementary to polynucleotidesencoding NHC. Thus, antisense sequences may be used to modulate NHCactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding NHC.

[0130] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding NHC. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra).

[0131] Genes encoding native NHC can be turned off by transforming acell or tissue with expression vectors which express high levels of thepolynucleotide, or fragment thereof, which encodes NHC. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the genomic DNA,such vectors may continue to transcribe RNA molecules until they aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

[0132] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding NHC, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0133] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding NHC.

[0134] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0135] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding NHC. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0136] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0137] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0138] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0139] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of NHC,antibodies to NHC, mimetics, agonists, antagonists, or inhibitors ofNHC. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0140] The pharmaceutical compositions utilized ill this invention maybe administered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intraperitoneal, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0141] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0142] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0143] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0144] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0145] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0146] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks's solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0147] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0148] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0149] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0150] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of NHC, such labeling wouldinclude amount, frequency, and method of administration.

[0151] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0152] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0153] A therapeutically effective dose refers to that amount of activeingredient, for example NHC or fragments thereof, antibodies of NHC,agonists, antagonists or inhibitors of NHC, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0154] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0155] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0156] Diagnostics

[0157] In another embodiment, antibodies which specifically bind NHC maybe used for the diagnosis of conditions or diseases characterized byexpression of NHC, or in assays to monitor patients being treated withNHC, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for NHC includemethods which utilize the antibody and a label to detect NHC in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0158] A variety of protocols including ELISA, RIA, and FACS formeasuring NHC are known in the art and provide a basis for diagnosingaltered or abnormal levels of NHC expression. Normal or standard valuesfor NHC expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to NHC under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of NHCexpressed in control and disease, samples from biopsied tissues arecompared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0159] In another embodiment of the invention, the polynucleotidesencoding NHC may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNHC may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of NHC, andto monitor regulation of NHC levels during therapeutic intervention.

[0160] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NHC or closely related molecules, may be used to identifynucleic acid sequences which encode NHC. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding NHC, alleles, or related sequences.

[0161] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the sequences encoding NHC. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequences ofSEQ ID NO:2 and SEQ ID NO:5 or from genomic sequence including promoter,enhancer elements, and introns of the naturally occurring NHC.

[0162] Means for producing specific hybridization probes for DNAsencoding NHC include the cloning of nucleic acid sequences encoding NHCor NHC derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0163] Polynucleotide sequences encoding NHC may be used for thediagnosis of conditions or diseases which are associated with expressionof NHC. Examples of such conditions or diseases include cancers,inflammation, and conditions affecting pregnancy, growth, anddevelopment as previously described. The polynucleotide sequencesencoding NHC may be used in Southern or northern analysis, dot blot, orother membrane-based technologies; in PCR technologies; or in dip stick,pin, ELISA or chip assays utilizing fluids or tissues from patientbiopsies to detect altered NHC expression. Such qualitative orquantitative methods are well known in the art.

[0164] In a particular aspect, the nucleotide sequences encoding NHC maybe useful in assays that detect activation or induction of variouscancers, particularly those mentioned above . The nucleotide sequencesencoding NHC may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding NHC in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0165] In order to provide a basis for the diagnosis of diseaseassociated with expression of NHC, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes NHC, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0166] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0167] With respect to cancer, the presence of a relatively low amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0168] Additional diagnostic uses for oligonucleotides designed from thesequences encoding NHC may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′←3′ ) and another withantisense (3′→5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0169] Methods which may also be used to quantitate the expression ofNHC include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0170] In another embodiment of the invention, the nucleic acid sequencewhich encodes NHC may also be used to generate hybridization probeswhich are useful for mapping the naturally occurring genomic sequence.The sequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques . Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0171] FISH (as described in Verma, R. S. et al. (1988) HumanChromosomes: A Manual of Basic Techniques, Pergamon Press, New York,N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in the 1994 Genome Issue of Science (265:1981f). Correlationbetween the location of the gene encoding NHC on a physical chromosomalmap and a specific disease, or predisposition to a specific disease, mayhelp delimit the region of DNA associated with that genetic disease. Thenucleotide sequences of the subject invention may be used to detectdifferences in gene sequences between normal, carrier, or affectedindividuals.

[0172] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0173] In another embodiment of the invention, NHC, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between NHCand the agent being tested, may be measured.

[0174] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to NHC large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with NHC, or fragments thereof, and washed. Bound NHC is thendetected by methods well known in the art. Purified NHC can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0175] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding NHCspecifically compete with a test compound for binding NHC. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with NHC.

[0176] In additional embodiments, the nucleotide sequences which encodeNHC may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0177] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

[0178] EXAMPLES

I SINTBST01 cDNA Library Construction and Isolation of cDNA Clones

[0179] The SINTBST01 cDNA library was constructed from the ileum of an18-year-old Caucasian female with irritable bowel syndrome. The patientpresented with abdominal pain and symptoms of enteritis. The patient wastreated with Priloxec® (omeprazole; Astra/Merck, Wayne, Pa.); Pentasa®(mesalamine;; Marion Merrell Dow, Kansas City, Mo.); and amoxicillin.The patient history included abnormal blood chemistry and osteoporosis,and family history included cerebrovascular and cardiovascular disease.

[0180] The frozen ileum tissue was homogenized and lysed in guanidiniumisothiocyanate solution using a Brinkmann POLYTRON homogenizer PT-3000(Brinkmann Instruments, Westbury, N.J.). The lysate was centrifuged overa 5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70Multracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.0,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. Extractionand precipitation were repeated as before. The mRNA was isolated withthe OLIGOTEX-kit (Qiagen, Inc., Chatsworth, Calif.) and used toconstruct the cDNA library.

[0181] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT cDNA synthesis and plasmid cloning system (Cat. No.18248-013, GIBCO/BRL, Gaithersburg, Md.). cDNAs were fractionated on aSEPHAROSE CL4B column (Cat. No. 275105-01, Pharmacia Upjohn), and thosecDNAs exceeding 400 bp were ligated into pINCY. The plasmid wassubsequently transformed into DH5α competent cells (Cat. No.18258-012,GIBCO/BRL, Gaithersburg, Md.).

[0182] Plasmid or phagemid DNA was released from cells and purifiedusing the Miniprep Kit (Cat. No. 77468; Advanced Genetic TechnologiesCorporation, Gaithersburg Md.). This kit consists of a 96 well blockwith reagents for 960 purifications. The recommended protocol wasemployed except for the following changes: 1) the 96 wells were eachfilled with only 1 mil of sterile Terrific Broth (Cat. No. 22711,GIBCO/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) thebacteria were cultured for 24 hours after the wells were inoculated andthen lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R at 2900 rpm for 5 min was performed beforethe contents of the block were added to the primary filter plate; and 4)the optional step of adding isopropanol to Tris buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

[0183] Alternative methods of purifying plasmid DNA include the use ofMAGIC MINIPREPS DNA purification system (Cat. No. A7 100, Promega) orQIAWELL-8 Plasmid, QIAWELL PLUS DNA and QIAWELL ULTRA DNA purificationsystems (Qiagen, Inc.).

[0184] II OVARTUT01 cDNA Library Construction and Isolation of cDNAClones

[0185] The OVARTUT01 cDNA library was constructed from tumorous ovarytissue obtained from a 43 year old Caucasian female with a malignantneoplasm The patient history indicated a previous normal delivery and avaginal hysterectomy. Also reported in the patient history were previousdiagnoses of hepatitis, cerebrovascular disease, atherosclerosis andmitral valve disorder; however, the patient was not taking medicationfor any of these conditions at the time of surgery.

[0186] The tissue was processed and cDNAs were made as described. ThecDNAs were ligated into PSPORT I which was subsequently transformed intoDH5α competent cells (Cat. #18258-012; GIBCO/BRL).

[0187] Plasmid DNA was released from the cells and purified using theREAL PREP 96-well plasmid isolation kit (Catalog #26173; Qiagen, Inc.).This kit enables the simultaneous purification of 96 samples in a96-well block using multi-channel reagent dispensers. The recommendedprotocol was employed except for the following changes:1) the bacteriawere cultured in 1 ml of sterile Terrific Broth (Catalog #22711,GIBCO/BRL) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) thecultures were incubated for 19 hours after the wells were inoculated andthen lysed with 0.3 ml of lysis buffer; 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a Beckman 96-well block for storage.

[0188] The cDNAs for both SINTBST01 and OVARTUT01 libraries weresequenced by the method of Sanger F and AR Coulson (1975; J Mol Biol94:441f), using a Hamilton MICROLAB 2200 (Hamilton, Reno Nev.) incombination with Peltier Thermal cyclers (PTC200 from MJ Research,Watertown Ma.) and Applied Biosystems 377 or 373 DNA sequencing systems;and the reading frame was determined.

[0189] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0190] The nucleotide sequences of the Sequence Listing or amino acidsequences deduced from them were used as query sequences againstdatabases such as GenBank, SwissProt, BLOCKS, and Pima II. Thesedatabases which contain previously identified and annotated sequenceswere searched for regions of homology (similarity) using BLAST, whichstands for Basic Local Alignment Search Tool (Altschul (1993) supra,Altschul (1990) supra).

[0191] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992;Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the sequences havelengths of at least 49 nucleotides, and no more than 12% uncalled bases(where N is recorded rather than A, C, G, or T).

[0192] The BLAST approach, as detailed in Karlin and Altschul (supra)and incorporated herein by reference, searches for matches between aquery sequence and a database sequence, to evaluate the statisticalsignificance of any matches found, and to report only those matcheswhich satisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

[0193] Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology. The relevantdatabase for a particular match were reported as a GIxxx±p (where xxx ispri, rod, etc and if present, p=peptide) as shown in Table 1. In column3 of Table 1, the product score is calculated as follows: the %nucleotide or amino acid identity [between the query and referencesequences] in BLAST is multiplied by the % maximum possible BLAST score[based on the lengths of query and reference sequences] and then dividedby 100. Where an Incyte Clone was homologous to several sequences, up tofive matches were provided with their relevant scores. In an analogy tothe hybridization procedures used in the laboratory, the electronicstringency for an exact match was set at 70, and the conservative lowerlimit for an exact match was set at approximately 40 (with 1-2% errordue to uncalled bases). Column 4 provides the log-likelihood where thevalue reflects the log of (probability divided by threshold); column 5,the relevant GenBank release; and column 6, a GenBank description of theprotein, or an edited version thereof. Some of the GenBank descriptionspresented in the tables of this application were standardized withrespect to abbreviations and spelling.

[0194] IV Northern Analysis

[0195] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0196] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesill nucleotide databases such as GenBank or the LIFESEQ™ database(Incyte Pharmaceuticals). This analysis is much faster than multiple, ismembrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0197] The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score 100

[0198] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0199] The results of northern analysis are reported as a list oflibraries in which the transcript encoding NHC occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0200] V Extension of Polynucleotides Encoding NHC to Full Length or toRecover Regulatory Sequences

[0201] Polynucleotides encoding NHC (SEQ ID NO:2 and SEQ ID NO:5) areused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating Applic containing new, unknown nucleotidesequence for the region of interest. The initial primers are designedfrom the cDNA using OLIGO 4.06 primer analysis software (NationalBiosciences), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationsis avoided.

[0202] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known. region.

[0203] By following the instructions for the XL-PCR kit (AppliedBiosystems) and thoroughly mixing the enzyme and reaction mix, highfidelity amplification is obtained. Beginning with 40 pmol of eachprimer and the recommended concentrations of all other components of thekit, PCR is performed using the Peltier Thermal cycler (PTC200; M.J.Research, Watertown, Ma.) and the following parameters: Step 1 94° C.for 1 min (initial denaturation) Step 2 65° C. for 1 min Step 3 68° C.for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C.for 7 min Step 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C.for 15 sec Step 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11Repeat step 8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C.(and holding)

[0204] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK DNA purification kit (Qiagen Inc.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0205] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2×Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2×Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0206] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0207] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0208] VI Labeling and Use of Hybridization Probes

[0209] Hybridization probes derived from SEQ ID NO:2 and SEQ ID NO:5 areemployed to screen cDNAs, genomic DNAs, or mRNAs. Although the labelingof oligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 primer analysis software (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μC.i of [γ-³²P]adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN, Boston, Ma.). The labeled oligonucleotides are substantiallypurified with SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn).A portion containing 10⁷ counts per minute of each of the sense andantisense oligonucleotides is used in a typical membrane basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN).

[0210] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (NYTRAN PLUS membrane, Schleicher& Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at40° C. To remove nonspecific signals, blots are sequentially washed atroom temperature under increasingly stringent conditions up to0.1×saline sodium citrate and 0.5% sodium dodecyl sulfate. After XOMATAR autoradiography film (Kodak, Rochester, N.Y.) is exposed to theblots, or the blots are placed in a PHOSPHOIMAGER cassette (MolecularDynamics, Sunnyvale, Calif.) for several hours, hybridization patternsare compared visually.

[0211] VII Antisense Molecules

[0212] Antisense molecules to the sequence encoding NHC, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring NHC. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the sequences encoding NHC is used to inhibit expression of naturallyoccurring NHC. The complementary oligonucleotide is designed from themost unique 5′ sequence as shown in and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of a transcript encoding NHC bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5′ sequence of SEQ ID NO:2 and SEQ ID NO:5, an effectiveantisense oligonucleotide includes any 15-20 nucleotides spanning theregion which translates into the signal or 5′ coding sequence of thepolypeptide as shown in FIGS. 1 and 5.

[0213] VIII Expression of NHC

[0214] Expression of NHC is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORT, previously used for thegeneration of the cDNA library is used to express NHC in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0215] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of NHC into the bacterial growth media which can be useddirectly in the following assay for activity.

[0216] IX Demonstration of NHC Activity

[0217] Demonstration of NHC-1 activity is assayed by adding the NHC-1 ora vector expressing NHC-1 to cultured NIH3T3 cells. The differencesbetween rates of cell proliferation in treated versus untreated cellscan be monitored either microscopically or spectrophotometrically.

[0218] Demonstration of NHC-2 activity is assayed in a Lewis to F344 ratmodel of heterotopic cardiac rejection as described in Utans, U. et al.(supra).

[0219] X Production of NHC Specific Antibodies

[0220] NHC that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 and SEQ ID NO:5 is analyzed usingDNASTAR software (DNASTAR Inc.) to determine regions of highimmunogenicity and a corresponding oligopolypeptide is synthesized andused to raise antibodies by means known to those of skill iii the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

[0221] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems 431 A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimnide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0222] XI Purification of Naturally Occurring NHC Using SpecificAntibodies

[0223] Naturally occurring or recombinant NHC is substantially purifiedby immunoaffinity chromatography using antibodies specific for NHC. Animmunoaffinity column is constructed by covalently coupling NHC antibodyto an activated chromatographic resin, such as CnBr-activated SEPHAROSE(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0224] Media containing NHC is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of NHC (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NHC binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and NHC is collected.

[0225] XII Identification of Molecules Which Interact with NHC

[0226] NHC or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973)Biochem. J. 133:529-39). Candidate molecules previously arrayed in thewells of a multi-well plate are incubated with the labeled NHC, washedand any wells with labeled NHC complex are assayed. Data obtained usingdifferent concentrations of NHC are used to calculate values for thenumber, affinity, and association of NHC with the candidate molecules.

[0227] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 7 69 amino acids amino acid single linear SINTBST01 1431384 1 Met TyrIle Thr Ala Gln Gly Val Asn Ser Ala Leu Gln Cys Ser Phe 1 5 10 15 SerGln Phe Tyr Ser Ala Val Leu Val Ser Phe Ser Cys Ile Gly Phe 20 25 30 HisCys Ile Tyr Ser Leu Phe Met Leu Asn Leu Ala Lys Asp Glu His 35 40 45 CysPro Pro Leu Lys Cys Leu Cys His Phe Glu Phe Cys Ala Thr Phe 50 55 60 ValAla Arg Met Arg 65 482 base pairs nucleic acid single linear SINTBST011431384 2 GAATTAAATG CAGCAGGCTT TATTTTAAAT GCCGATTCAC ATTACTCTGTTCAAGCTGC 60 TTGAGATGTT AAACTGGCTT ACTATAGACT TCGTAAAAAT GGCTCCAGAAGAGTAACA 120 CTGAAATCTT TGAGATCACA CAGGTTGGAA ATATGTACAT AACTGCACAAGGTGTCAA 180 CTGCTCTACA GTGCAGTTTT AGTCAGTTTT ACAGTGCAGT TTTAGTCAGTTTTAGTTG 240 TAGGTTTCCA TTGTATTTAT AGTCTGTTTA TGCTAAATCT GGCCAAAGATGAGCATTG 300 CACCACTAAA ATGCCTCTGC CACTTTGAAT TCTGTGCTAC TTTTGTGGCCAGAATGCG 360 GATCAAAACG CTCCATCTTT TTACAGTGGC ATAGGAAGAC GGCAAAAATTTCCTAAAG 420 CAATAGATTT TCAAGTGTAT TGTGCCTTGT TCTAAAACTT TTATTAAGTAGGTGCCTT 480 CA 482 61 amino acids amino acid single linear GenBank1353711 3 Met Tyr Ile Thr Glu Gln Gly Val Asn Ser Ala Leu Gln Cys SerLeu 1 5 10 15 Val Ser Phe Ser Cys Ile Gly Phe His Cys Phe Tyr Ser LeuPhe Met 20 25 30 Leu Asn Leu Ala Lys Asp Glu His Cys Pro Pro Leu Lys CysLeu Cys 35 40 45 His Trp Glu Phe Trp Val Asn Phe Val Thr Arg Met Gln 5055 60 147 amino acids amino acid single linear OVARTUT01 815614 4 MetSer Gln Thr Arg Asp Leu Gln Gly Gly Lys Ala Phe Gly Leu Leu 1 5 10 15Lys Ala Gln Gln Glu Glu Arg Leu Asp Glu Ile Asn Lys Gln Phe Leu 20 25 30Asp Asp Pro Lys Tyr Ser Ser Asp Glu Asp Leu Pro Ser Lys Leu Glu 35 40 45Gly Phe Lys Glu Lys Tyr Met Glu Phe Asp Leu Asn Gly Asn Gly Asp 50 55 60Ile Asp Ile Met Ser Leu Lys Arg Met Leu Glu Lys Leu Gly Val Pro 65 70 7580 Lys Thr His Leu Glu Leu Lys Lys Leu Ile Gly Glu Val Ser Ser Gly 85 9095 Ser Gly Glu Thr Phe Ser Tyr Pro Asp Phe Leu Arg Met Met Leu Gly 100105 110 Lys Arg Ser Ala Ile Leu Lys Met Ile Leu Met Tyr Glu Glu Lys Ala115 120 125 Arg Glu Lys Glu Lys Pro Thr Gly Pro Pro Ala Lys Lys Ala IleSer 130 135 140 Glu Leu Pro 145 658 base pairs nucleic acid singlelinear OVARTUT01 815614 5 GGCTTCTGAG AAGACTGGNG GGAGAGAAGG AGAGCCTGCAGACAGAGGCC TCCAGCTTG 60 TCTGTCTCCC CACCTCTACC AGCATCTGCT GAGCTATGAGCCAAACCAGG GATTTACA 120 GAGGAAAAGC TTTCGGACTG CTGAAGGCCC AGCAGGAAGAGAGGCTGGAT GAGATCAA 180 AGCAATTCCT AGACGATCCC AAATATAGCA GTGATGAGGATCTGCCCTCC AAACTGGA 240 GCTTCAAAGA GAAATACATG GAGTTTGACC TTAATGGAAATGGCGATATT GATATCAT 300 CCCTGAAACG AATGCTGGAG AAACTTGGAG TCCCCAAGACTCACCTAGAG CTAAAGAA 360 TAATTGGAGA GGTGTCCAGT GGCTCCGGGG AGACGTTCAGCTACCCTGAC TTTCTCAG 420 TGATGCTGGG CAAGAGATCT GCCATCCTAA AAATGATCCTGATGTATGAG GAAAAAGC 480 GAGAAAAGGA AAAGCCAACA GGCCCCCCAG CCAAGAAAGCTATCTCTGAG TTGCCCTG 540 TTGAAGGGAA AAGGGATGAT GGGATTGAAG GGGCTTCTAATGACCCAGAT ATGGAAAC 600 AAGACAAAAT TGTAAGCCAG AGTCAACAAA TTAAATAAATTACCCCCTCC TCCAGATC 658 147 amino acids amino acid single linear GenBank1122909 6 Met Ser Gln Thr Arg Asp Leu Gln Gly Gly Lys Ala Phe Gly LeuLeu 1 5 10 15 Lys Ala Gln Gln Glu Glu Arg Leu Asp Glu Ile Asn Lys GlnPhe Leu 20 25 30 Asp Asp Pro Lys Tyr Ser Ser Asp Glu Asp Leu Pro Ser LysLeu Glu 35 40 45 Gly Phe Lys Glu Lys Tyr Met Glu Phe Asp Leu Asn Gly AsnGly Asp 50 55 60 Ile Asp Ile Met Ser Leu Lys Arg Met Leu Glu Lys Leu GlyVal Pro 65 70 75 80 Lys Thr His Leu Glu Leu Lys Lys Leu Ile Gly Glu ValSer Ser Gly 85 90 95 Ser Gly Glu Thr Phe Ser Tyr Pro Asp Phe Leu Arg MetMet Leu Gly 100 105 110 Lys Arg Ser Ala Ile Leu Lys Met Ile Leu Met TyrGlu Glu Lys Ala 115 120 125 Arg Glu Lys Glu Lys Pro Thr Gly Pro Pro AlaLys Lys Ala Ile Ser 130 135 140 Glu Leu Pro 145 146 amino acids aminoacid single linear GenBank 1514969 7 Met Lys Pro Glu Glu Ile Ser Arg GlyLys Ala Phe Gly Leu Leu Lys 1 5 10 15 Ala Gln Gln Glu Glu Arg Leu AspGly Ile Asn Lys His Phe Leu Asp 20 25 30 Asp Pro Lys Tyr Ser Ser Asp GluAsp Leu Gln Ser Lys Leu Glu Ala 35 40 45 Phe Lys Thr Lys Tyr Met Glu PheAsp Leu Asn Gly Asn Gly Asp Ile 50 55 60 Asp Ile Met Ser Leu Lys Arg MetLeu Glu Lys Leu Gly Val Pro Lys 65 70 75 80 Thr His Leu Glu Leu Lys LysLeu Ile Arg Glu Val Ser Ser Gly Ser 85 90 95 Glu Glu Thr Phe Ser Tyr SerAsp Phe Leu Arg Met Met Leu Gly Lys 100 105 110 Arg Ser Ala Ile Leu ArgMet Ile Leu Met Tyr Glu Glu Lys Asn Lys 115 120 125 Glu His Gln Lys ProThr Gly Pro Pro Ala Lys Lys Ala Ile Ser Glu 130 135 140 Leu Pro 145

What is claimed is:
 1. An isolated polypeptide selected from the group consisting of: a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 2. An isolated polypeptide of claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 3. An isolated polynucleotide encoding a polypeptide of claim
 1. 4. An isolated polynucleotide encoding a polypeptide of claim
 2. 5. An isolated polynucleotide of claim 4 comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5.
 6. A recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide of claim
 3. 7. A cell transformed with a recombinant polynucleotide of claim
 6. 8. A transgenic organism comprising a recombinant polynucleotide of claim
 6. 9. A method of producing a polypeptide of claim 1, the method comprising: a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide, and said recombinant polynucleotide comprises a promoter sequence operably linked to a polynucleotide encoding the polypeptide of claim 1, and b) recovering the polypeptide so expressed.
 10. A method of claim 9, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 11. An isolated antibody which specifically binds to a polypeptide of claim
 1. 12. An isolated polynucleotide selected from the group consisting of: a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:5, c) a polynucleotide complementary to a polynucleotide of a), d) a polynucleotide complementary to a polynucleotide of b), and e) an RNA equivalent of a)-d).
 13. An isolated polynucleotide comprising at least 60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
 15. A method of claim 14, wherein the probe comprises at least 60 contiguous nucleotides.
 16. A method of detecting a target polynucleotide in a sample, said target polynucleotide having a sequence of a polynucleotide of claim 12, the method comprising: a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
 17. A composition comprising a polypeptide of claim 1 and a pharmaceutically acceptable excipient.
 18. A composition of claim 17, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 19. A method for treating a disease or condition associated with decreased expression of functional NHC, comprising administering to a patient in need of such treatment the composition of claim
 17. 20. A method of screening a compound for effectiveness as an agonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting agonist activity in the sample.
 21. A composition comprising an agonist compound identified by a method of claim 20 and a pharmaceutically acceptable excipient.
 22. A method for treating a disease or condition associated with decreased expression of functional NHC, comprising administering to a patient in need of such treatment a composition of claim
 21. 23. A method of screening a compound for effectiveness as an antagonist of a polypeptide of claim 1, the method comprising: a) exposing a sample comprising a polypeptide of claim 1 to a compound, and b) detecting antagonist activity in the sample.
 24. A composition comprising an antagonist compound identified by a method of claim 23 and a pharmaceutically acceptable excipient.
 25. A method for treating a disease or condition associated with overexpression of functional NHC, comprising administering to a patient in need of such treatment a composition of claim
 24. 26. A method of screening for a compound that specifically binds to the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide of claim 1 to the test compound, thereby identifying a compound that specifically binds to the polypeptide of claim
 1. 27. A method of screening for a compound that modulates the activity of the polypeptide of claim 1, the method comprising: a) combining the polypeptide of claim 1 with at least one test compound under conditions permissive for the activity of the polypeptide of claim 1, b) assessing the activity of the polypeptide of claim 1 in the presence of the test compound, and c) comparing the activity of the polypeptide of claim 1 in the presence of the test compound with the activity of the polypeptide of claim 1 in the absence of the test compound, wherein a change in the activity of the polypeptide of claim 1 in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide of claim
 1. 28. A method of screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a sequence of claim 5, the method comprising: a) exposing a sample comprising the target polynucleotide to a compound, under conditions suitable for the expression of the target polynucleotide, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
 29. A method of assessing toxicity of a test compound, the method comprising: a) treating a biological sample containing nucleic acids with the test compound, b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of claim 12 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of claim 12 or fragment thereof, c) quantifying the amount of hybridization complex, and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
 30. A method for a diagnostic test for a condition or disease associated with the expression of NHC in a biological sample, the method comprising: a) combining the biological sample with an antibody of claim 11, under conditions suitable for the antibody to bind the polypeptide and form an antibody:polypeptide complex, and b) detecting the complex, wherein the presence of the complex correlates with the presence of the polypeptide in the biological sample.
 31. The antibody of claim 11, wherein the antibody is: a) a chimeric antibody, b) a single chain antibody, c) a Fab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 32. A composition comprising an antibody of claim 11 and an acceptable excipient.
 33. A method of diagnosing a condition or disease associated with the expression of NHC in a subject, comprising administering to said subject an effective amount of the composition of claim
 32. 34. A composition of claim 32, wherein the antibody is labeled.
 35. A method of diagnosing a condition or disease associated with the expression of NHC in a subject, comprising administering to said subject an effective amount of the composition of claim
 34. 36. A method of preparing a polyclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibodies from the animal, and c) screening the isolated antibodies with the polypeptide, thereby identifying a polyclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 37. A polyclonal antibody produced by a method of claim
 36. 38. A composition comprising the polyclonal antibody of claim 37 and a suitable carrier.
 39. A method of making a monoclonal antibody with the specificity of the antibody of claim 11, the method comprising: a) immunizing an animal with a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4, or an immunogenic fragment thereof, under conditions to elicit an antibody response, b) isolating antibody producing cells from the animal, c) fusing the antibody producing cells with immortalized cells to form monoclonal antibody-producing hybridoma cells, d) culturing the hybridoma cells, and e) isolating from the culture monoclonal antibody which specifically binds to a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 40. A monoclonal antibody produced by a method of claim
 39. 41. A composition comprising the monoclonal antibody of claim 40 and a suitable carrier.
 42. The antibody of claim 11, wherein the antibody is produced by screening a Fab expression library.
 43. The antibody of claim 11, wherein the antibody is produced by screening a recombinant immunoglobulin library.
 44. A method of detecting a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4 in a sample, the method comprising: a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) detecting specific binding, wherein specific binding indicates the presence of a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4 in the sample.
 45. A method of purifying a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4 from a sample, the method comprising: a) incubating the antibody of claim 11 with the sample under conditions to allow specific binding of the antibody and the polypeptide, and b) separating the antibody from the sample and obtaining the purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:4.
 46. A microarray wherein at least one element of the microarray is a polynucleotide of claim
 13. 47. A method of generating an expression profile of a sample which contains polynucleotides, the method comprising: a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray of claim 46 with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
 48. An array comprising different nucleotide molecules affixed in distinct physical locations on a solid substrate, wherein at least one of said nucleotide molecules comprises a first oligonucleotide or polynucleotide sequence specifically hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, and wherein said target polynucleotide is a polynucleotide of claim
 12. 49. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 30 contiguous nucleotides of said target polynucleotide.
 50. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to at least 60 contiguous nucleotides of said target polynucleotide.
 51. An array of claim 48, wherein said first oligonucleotide or polynucleotide sequence is completely complementary to said target polynucleotide.
 52. An array of claim 48, which is a microarray.
 53. An array of claim 48, further comprising said target polynucleotide hybridized to a nucleotide molecule comprising said first oligonucleotide or polynucleotide sequence.
 54. An array of claim 48, wherein a linker joins at least one of said nucleotide molecules to said solid substrate.
 55. An array of claim 48, wherein each distinct physical location on the substrate contains multiple nucleotide molecules, and the multiple nucleotide molecules at any single distinct physical location have the same sequence, and each distinct physical location on the substrate contains nucleotide molecules having a sequence which differs from the sequence of nucleotide molecules at another distinct physical location on the substrate.
 56. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:1.
 57. A polypeptide of claim 1, comprising the amino acid sequence of SEQ ID NO:4
 58. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:2.
 59. A polynucleotide of claim 12, comprising the polynucleotide sequence of SEQ ID NO:5. 